1. Technical Field
The present invention is in the field of bioreactors for cell culture. More specifically, the invention is in the field of bioreactors which allow for flow-through of media while retaining non-adherent as well as adherent cells within the bioreactor chamber.
2. Related Technology
In cell culture, it is often desirable to maintain cells in vitro for an extended time, during which the cells produce waste, acidify the medium, and use up nutrients from the medium. The exhaustion of the medium is accelerated when the cells proliferate and/or differentiate into highly metabolic cell types. Thus a central problem in cell culture is providing a means to refresh the culture medium without disturbing the cells.
Cell types which adhere to the surface of a culture flask may have their media exchanged or refreshed by simply pouring off the spent media and pouring in fresh media. Alternatively, a portion of the spent media may be gently drawn off and replaced with fresh media. Perfusion or flow-through of fresh media may be desirable for the growth of adherent cell types which require frequent or constant refreshment of culture media. However, even adherent cells may be adversely affected by the shear stress inflicted by the bulk flow of media. Adherent cells may be forced away from their moorings by the bulk media flow, and then lost from the culture system. Alternatively, adherent cells may stay attached to their substrate, but be adversely affected by the force of the fluid such that they fail to proliferate and/or differentiate. Part of the adverse effects of perfusion cultures may be attributed to dilution and wash out of factors produced by the cells themselves, when those factors are necessary for cell development.
Cell types which do not adhere to surfaces, but rather grow in suspension, present an extra challenge for media exchange. The problem is to exchange the media without losing a high proportion of the cells in the spent media.
Non-adherent cells may be retained in bioreactors with the use of physical barriers. A physical barrier may be in the form of a membrane that creates a barrier to the passage of cells, but allows the diffusion of nutrients and metabolic byproducts.
Hollow fiber bioreactors work on the principle of physical barriers. In a hollow fiber bioreactor, the cells are retained behind a semi-permeable membrane (i.e., the fiber material). A typical hollow fiber unit contains thousands of individual hollow fibers. Commonly, the cells are cultured in the spaces surrounding the fibers. Culture media is perfused through the spaces, and metabolic byproducts diffuse through the semi-permeable membrane, into the hollow fibers, and then out of the system. Examples of hollow fiber bioreactors are disclosed in WO 91/18972 (Knazek) and WO 92/10564 (Culver).
Other types of bioreactors are based on the use of semi-permeable membranes or supports (U.S. Pat. No. 5,264,344 (Sneath) and U.S. Pat. No. 5,223,428 (Rose).
The roller-bottle type of bioreactor is designed for even distribution of medium throughout the cell population. Traditionally, cells adhere to the inner surface of the bottle, which is constantly rotated to bathe the cells. Certain roller-bottle bioreactors have increased inner surface area provided by support strips or corrugations (U.S. Pat. No. 5,010,013 (Serkes); EP 345 415 (Tyndorf); U.S. Pat. No. 3,853,712; U.S. Pat. No. 5,270,205 (Rogalsky); U.S. Pat. No. 5,256,570 (Clyde)).
Other types of bioreactors, known as stirred bioreactors, often include the use of spin-filters and settling tubes in order to retain cells (U.S. Pat. No. 4,760,028 (deBruyne); U.S. Pat. No. 4,906,577 (Armstrong)). Anchorage-dependent cells may be grown on microcarrier beads, which are commonly used in stirred bioreactors (EP 046,681 (Tolbert); U.S. Pat. No. 5,002,890 (Morrison)).
Several types of static culture flasks make use of corrugations, ridges, or bristles on their internal surfaces in order to provide increased surface area for the growth of anchorage dependent cells (U.S. Pat. No. 5,084,393 (Rogalsky); U.S. Pat. No. 5,272,084 (O'Connell); U.S. Pat. No. 5,151,366 (Serkes)). U.S. Pat. No. 4,939,151 (Bacehowski) discloses a cell culture bag having a non-smooth inner surface to prevent the inner surfaces from sticking together during manufacturing and sterilization processes. A three-dimensional solid matrix has also been proposed for growing adherent cells (U.S. Pat. No. 4,514,499 (Noll).
Researchers have had the most experience to date culturing certain specific types of cells, including bacteria, antibody producing hybridomas, fibroblasts, and eukaryotic cell lines. Other types of cells, such as hematopoietic cells, present unusual challenges in the design of a suitable bioreactor.
For certain cancer treatments, it is desirable to culture hematopoietic cells in order to administer the cultured cells to a patient. Hematopoietic cells are obtained from a donor's or a patient's bone marrow or peripheral blood.
The starting cell suspension to be cultured may contain a variety of hematopoietic cells in various stages of differentiation. Alternatively, the cell suspension may first be subjected to certain selection processes, resulting in a starting cell sample highly enriched for stem cells, for instance. Stem cells are primitive hematopoietic cells which have the potential to differentiate into cells of all hematopoietic lineages, including granulocytes, lymphocytes, erythrocytes, and megakaryocytes. It is generally believed that stem cells require adherence to a substrate in order to proliferate and develop to a progenitor stage. However, the cells that have progressed to the progenitor stage, and beyond, are thought to be generally non-adherent because their in vivo micro-environment would be a moving fluid (blood), and they would not be adapted for adherence to a static surface. Thus a culture of hematopoietic cells may contain a variety of different cell types including adherent and non-adherent cells. To further complicate the picture, some of the non-adherent cells may adhere to other cells which, in turn, adhere to a surface.
Hematopoeitic cells present additional challenges because they are shear sensitive. Hematopoietic cells do not appear to grow well when suspended in spinner flask cultures. In attempts to provide a micro-evironment conducive to hematopoietic cell growth, growth surfaces have been provided with stromal layers. The stromal layer is generally selected to mimic the extracellular matrix in the bone marrow and consists of proteins such as collagen and fibronectin. Bioreactors which depend on the use of stroma are disclosed in WO 90/15877 (Emerson), WO 92/11355 (Emerson), EP 0 358 506 (Naughton), U.S. Pat. No. 5,160,490 (Naughton), and U.S. Pat. No. 4,963,489 (Naughton).
The use of stroma is disadvantageous for several reasons. First, it is time consuming to produce the stromal layer on a cell culture surface, and great care must be taken not to introduce contaminants into the culture vessels. Certain techniques for laying down stroma require the use of living cells, such as fibroblasts, which are different from the cell type to be cultured. The introduction of foreign cell types into a culture vessel complicates the task of culturing a hematopoietic cell suspension suitable for clinical use.
Accordingly, a primary object for this invention is to provide a bioreactor which allows for the exchange of media without undue perturbation or loss of the cultured cells.
Another object for this invention is to provide a bioreactor which permits retention of cells without the use of stroma.
Another object of this invention is to provide a flow-through bioreactor which permits cultured cells to be easily and efficiently recovered from the bioreactor chamber.
These and other objects and advantages of the present invention will be apparent from a reading of the following detailed description of exemplary preferred embodiments of the invention, taken in conjunction with the appended drawing Figures, in which the same reference numeral refers to the same feature throughout the drawing Figures, or to features which are analogous in structure or function. Dimensions of the grooves are identified as X, Y, and Z. The longitudinal axis of the entire bioreactor vessel is identified as L.